WO2019180519A1

WO2019180519A1 - Method and kit for isolation of nucleic acids from a sample

Info

Publication number: WO2019180519A1
Application number: PCT/IB2019/051497
Authority: WO
Inventors: Jiujun CHENG
Original assignee: Metagenom Bio Holdings Pte Ltd.
Priority date: 2018-03-17
Filing date: 2019-02-25
Publication date: 2019-09-26

Abstract

The invention relates to a method for isolating nucleic acids from a sample, such as an environmental or soil sample. The method comprises the steps of: a. providing the sample; b. lysis of biological cells in the sample in a reaction solution comprising a detergent, a first pH buffer, a DNA desorption agent such as sodium phosphate, a chelating agent, sodium chloride and silica beads; c. isolating nucleic acids and dissolved contaminants from the sample in a supernatant; d. contacting the supernatant with a flocculant to precipitate and remove proteins; e. contacting the supernatant with a chaotropic agent and a second pH buffer; and f. purification of the nucleic acids by anion exchange on a spin column comprising a silica gel membrane. Said method can reduce the amount of contaminants that inhibit the efficiency of downstream applications, such as polymerase chain reaction (PCR).

Description

METHOD AND KIT FOR ISOLATION OF NUCLEIC ACI DS FROM A

SAMPLE

Field of the I nvention

The present invention relates to a method and kit for isolating nucleic acids, and more specifically, to a method and kit for isolating nucleic acids from a broad range of environmental and process samples.

Background Art

Methods and kits for isolating nucleic acids, such as deoxyribonucleic acid (DNA), from samples, such as environmental samples including soil, are known. Such methods and kits may be useful for detecting, analyzing, characterizing and cloning nucleic acid sequences of interest. For example, a DNA sequence of interest may be isolated and subsequently amplified by polymerase chain reaction (PCR). The efficient isolation and amplification of target nucleic acid sequences is useful in a wide variety of applications in a broad range of fields, including but not limited to agriculture, environmental and ecosystem sciences, molecular biology, plant biology, forensics, clinical diagnostics and gene therapy.

However, known methods and kits have a drawback in that contaminants are co-extracted with the nucleic acids, and inhibit the efficiency of downstream applications such as amplification by PCR. In environmental samples, for example, contaminants may include, but are not limited to, polyphenols, polysaccharides, humic acid, enzyme inhibitors, decomposing organic matter, plant cell wall components, chitin, plant pigments, fulvic acid, tannin and heavy metals. It is believed that such contaminants can make nucleic acids resistant to digestion by restriction endonucleases, inhibit Taq DNA polymerase which is an enzyme used in PCR and/or inhibit DNA-DNA hybridizations. As such, nucleic acid isolation and amplification for soil and sediment genomic analysis has met with limited success.

In view of the above, it would be beneficial to provide methods and kits for isolating nucleic acids from samples, while reducing the amount of contaminants that are co-extracted. A reduction in contaminants would im prove the quality of the isolated nucleic acids for downstream

applications such as PCR.

Su m m ary of the I nvention

The nucleic acid isolation method and kit of the present invention provides for rapid and efficient extraction of nucleic acids, such as m etagenom ic DNA, from a variety of samples. Cells, such as m icrobial cells in soil samples, are lysed by chem ical and m echanical m eans. Contam inants are rem oved from the resulting lysate. Nucleic acids are then purified using a spin colum n comprising a silica gel membrane. Preferably, no organic solvents or proteinases are used in this m ethod.

This method and kit have been validated by successful extraction of nucleic acids from soil sam ples, and PCR am plification followed by sequence analysis of m icrobial 16S ribosomal RNA (rRNA) and fungal internal transcribed spacer ( ITS) sequences.

I n one aspect, the present invention provides a method for isolating nucleic acids from a sam ple comprising the steps of: a. providing the sample; b. lysis of biological cells in the sam ple in a reaction solution, the reaction solution com prising a detergent, a first pH buffer, a chelating agent, a DNA desorption agent, sodium chloride and silica beads; c.

isolating nucleic acids and dissolved contam inants from the sample in a supernatant; d. contacting the supernatant with a flocculant to precipitate and rem ove proteins; e. contacting the supernatant with a chaotropic agent and a second pH buffer; and f. purification of the nucleic acids by anion exchange on a spin colum n comprising a silica gel m em brane.

Preferably, the purified nucleic acids from step f are substantially free of the contam inants which inhibit amplification of the nucleic acids by polymerase chain reaction ( PCR) .

I n another aspect, the present invention provides a nucleic acid isolation kit for conducting the method of the present invention. Preferably, the nucleic acid isolation kit comprises:

(i) one or more bead tubes for holding silica beads;

(ii) a reaction solution comprising a detergent, a first pH buffer, a chelating agent, a DNA desorption agent, sodium chloride and the silica beads;

(iii) a flocculant;

(iv) a chaotropic agent and a second pH buffer;

(v) one or more spin columns; and

(vi) one or more collection tubes.

Description of the Drawings

Figure 1 shows the gel electrophoresis results of DNA isolation from different soil samples using: (i) Sox™ DNA Isolation Kit (MBI, Metagenom Bio™ Inc.) (a preferred embodiment of the present invention); (ii)

PowerSoil™ Isolation Kit (MB, MO BIO™ Laboratories, Inc.); and (iii) Soil DNA Isolation Plus Kit (N, Norgen Biotek™ Corporation).

Figure 2 shows the UV-visible spectra of soil DNA preps extracted with a preferred kit of the present invention.

Figure 3 shows the 16S (A) and ITS1 (B) amplicons from metagenomic DNA isolated with: (i) Sox™ DNA Isolation Kit (MBI, Metagenom Bio™

Inc.) (a preferred embodiment of the present invention); (ii) PowerSoil™ Isolation Kit (MB, MO BIO™ Laboratories, Inc.); and (iii) Soil DNA Isolation Plus Kit (N, Norgen Biotek™ Corporation).

Figures 4A and 4B show soil community profiles from DNA extracted using a preferred kit of the present invention.

Figure 5 shows a preferred kit of the present invention along with its packaging.

Detailed Description of Preferred Embodiments

The present invention provides a method and kit for isolating nucleic acids, such as deoxyribonucleic acid (DNA), from a sample, such as a soil sample. I n the method of the present invention, nucleic acids are preferably recovered in a quantity and concentration suitable for amplification through polymerase chain reaction ( PCR) . The isolated nucleic acids are preferably free, or substantially free, of contam inants which may inhibit PCR. Preferred steps in the method of the present invention are described below.

A sam ple comprising biological cells with the nucleic acids is provided. The sample m ay comprise one or more of soil, sediment, sand, gravel, pebbles and com post. Preferably, the sample is an environmental or process sample, such as but not lim ited to: soil, gravel, m ining tailings, sm all pebbles, ore, silt, sedim ent, glacial till, sand from slow sand filtration apparatus, compost, rhizosphere soil, used inert hydroponic m edia such as rock wool, expanded clay pebbles, perlite and verm iculite, and coco coir.

Biological cells contained within the sample are lysed by chem ical and m echanical means in a reaction solution. The reaction solution preferably com prises a detergent, a first pH buffer, a chelating agent, a DNA desorption agent, sodium chloride and silica beads.

Preferably, the detergent is sodium dodecyl sulfate (SDS) .

Preferably, the first pH buffer com prises a com pound selected from the group consisting of Tris- HCI and sodium phosphate.

Preferably, the chelating agent is Ethylenediam inetetraacetic acid ( EDTA) .

Preferably, the DNA desorption agent is sodium phosphate.

I n one preferred aspect, the silica beads are glass beads having a diam eter of 0.1 m m and 0.5 m m . More preferably the 0.1 m m diam eter beads and the 0.5 m m bead are present in a ratio of about 3: 1 , or another ratio within 20% variance from 3: 1 . Nucleic acids and dissolved contam inants are separated from the bulk sample in the resulting supernatant, preferably by centrifuge.

Contam inants m ay include, but are not lim ited to, polyphenols,

polysaccharides, hum ic acid, enzyme inhibitors, decom posing organic m atter, plant cell wall components, chitin, plant pigments, fulvic acid, tannin and heavy metals.

The supernatant is contacted with a flocculant resulting in the precipitation and rem oval of proteins, preferably SDS-proteins. Preferably, the flocculant is a solution com prising acetate and potassium . More

preferably, the acetate and potassium are present in a m olar ratio of 5 Moles/ L (M) acetate: 3 Moles/ L (M) potassium or another m olar ratio within 20% variance from 5 Moles/ L (M) acetate: 3 Moles/ L (M) potassium .

The supernatant is contacted with a chaotropic agent and a second pH buffer.

Preferably, the chaotropic agent is selected from the group consisting of guanidine hydrochloride ( HCL) and guanidine (iso)thiocyanate.

Preferably, the second pH buffer is a buffer that provides an effective pH buffering range between pH 6.0 and pH 7.0. More preferably, the second pH buffer is 2-(N-morpholino) ethanesulfonic acid (MES) .

Nucleic acids are purified by anion exchange on a spin colum n comprising a silica gel m em brane.

I n one preferred embodiment, nucleic acids are isolated by alcohol precipitation. I n another preferred embodiment, nucleic acids are subject to phenol-chloroform extraction, followed by isolation through alcohol precipitation.

An exemplary protocol of a preferred em bodiment of the method and kit of the present invention is provided below. Materials provided in nucleic acid isolation kit

• Bead tubes: 2 ml screw-cap tubes with 0.5 grams of glass beads (0.1 mm and 0.5 mm diameter beads, preferably in ratio of 3:1)

• Solution Sox1 : 100 mM Tris-HCI (pH 8), 100 mM Na phosphate (pH 8), 50 mM EDTA (pH 8), 500 mM NaCI, 1% SDS

• Solution Sox2: 5 M acetate and 3 M potassium (mix 60 ml of 5 M potassium acetate, 11.5 ml glacial acetate acid and 28.5 ml H₂0)

• Solution Sox3: 5.5 M guanidine thiocyanate, 20 mM 2-(N- morpholino) ethanesulfonic acid), pH 6.5

• Solution Sox4: 10 mM Tris-HCI (pH 7.3), 80% ethanol

• Solution Sox5: 10 mM Tris-HCI, pH 8.5

• Spin columns with collection tubes (Angen™ Biotech, #51011)

The materials of the preferred kit of the present invention may be packaged as shown in Figure 5.

User Protocol

Kit reagents and components should be stored at room temperature. All procedures are performed at room temperature unless otherwise stated.

Solution Sox4 (concentrate) should be diluted with 56 ml of 96-100% ethanol before use, and kept away from open flames and sparks after adding ethanol.

Solution Sox3 should be kept away from light. Do not mix Solution Sox3 or extraction waste with bleach or any other form of acid.

If precipitate occurs in Solution Sox1 and Sox3, warm the solutions at 65°C with occasional mixing until the precipitate is completely dissolved.

1. Add 0.25 grams of soil sample into a provided bead tube.

2. Add 850 pi of Solution Soxl to the tube. Caution: If a sample has high water content, transfer the beads from bead tube to a 1.5 ml microcentrifuge tube (not provided), add wet sample to the empty bead tube, centrifuge at 12,000 x g for 5 minutes, and remove the liquid with a pipette tip. Transfer the glass beads back to the bead tube, and then add Solution Sox1.

3. Secure the tube horizontally on a flat-bed vortex pad with tape or on a vortex microtube holder. Vortex at maximal speed for 10 minutes. Alternatively, beat for 40 seconds at 6M/S using FastPrep™-24 instrument (MP Biomedicals) or other commercial bead beaters according to

recommendations from the manufacturer.

4. Centrifuge the tube at 12,000 x g for 5 minutes.

5. Transfer up to 550 pi of supernatant to a 1.5 ml microcentrifuge tube (not provided). If removal of RNA is required, add 10 mI of DNase- free RNase A (10 mg/ml, not provided) to the tube, mix by pipetting 3 times, and incubate for 10 minutes.

6. Add 55 mI of Solution Sox2 to the supernatant and mix by inverting the tube 5 times.

7. Incubate the tube on ice for 5 minutes.

8. Centrifuge the tube at 20,000 x g for 5 minutes.

9. Transfer 500 mI of supernatant, avoiding transfer of any of the pelleted material, to a clean 1.5 ml microcentrifuge tube (not provided). Caution: If the supernatant is unclear, transfer all solution to a new 1.5 ml microcentrifuge tube, centrifuge at 20,000 x g for 3 minutes to clarify the solution, and then transfer 500 mI of supernatant to a new 1.5 ml microcentrifuge tube.

10. Add 1000 mI of Solution Sox3 to the supernatant and mix by pipetting 3 times.

11. Load 600 mI solution into a spin column and centrifuge at 10,000 x g for 1 minute. Discard the flow through and add an additional 600 mI of solution to the spin column and centrifuge at 10,000 x g for 1 minute.

Load the remaining solution to the column and centrifuge at 10,000 x g for 1 minute.

12. Optional: if the sample has high content of humic acids, add 600 mI of Solution Sox3, wait for 2 minutes and centrifuge at 14,000 x g for 1 minute. Discard the flow through. Repeat this step once.

13. Add 600 mI of Solution Sox4, wait for 2 minutes and centrifuge at 14,000 x g for 1 minute. Discard the flow through. Repeat this step once. 14. Centrifuge the colum n in the collection tube at 14,000 x g for 2 m inutes.

15. Place the colum n in a 1 .5 m l m icrocentrifuge tube (not provided) .

16. Add 100 pi of Solution Sox5 to the center of white m em brane.

17. Wait for 2 m inutes and then centrifuge at 10,000 x g for 1 m inute.

18. Discard the spin colum n. The DNA is now ready for any downstream application

Assessing DNA yield

The amount of DNA can be determ ined by a NanoDrop spectrophotom eter using Solution Sox5 as a blank. Agarose gel electrophoresis can be employed to verify the DNA content and evaluate DNA shearing and/or degradation. I n addition, fluorom etric quantification of DNA can be used if m etagenom ic DNA sequencing is desired.

DNA storage

Solution Sox5 does not contain EDTA. It is recom m ended to store DNA prep frozen at -20° to -80°C. Com parison with other nucleic acid isolation kits

Different types of soil sam ples were used to com pare DNA isolation by three different kits, including a preferred embodim ent of the kit of the present invention. The physiochem ical properties of the soil samples is provided in Table 1 below. As can be seen, there is a wide range of such properties between different sam ples.

Table 1. Physiochemical properties of soil samples used for metagenomic DNA extraction

Sample Total Gravel Sand Silt [%] Clay Moisture name Carbon (%) (%) ( %) (% dry)

(% dryl

Tar Sands 2.76 0.00 80.20 14.40 5.40 16.15

Temperate 10.80 11.20 63.40 31.10 5.40 69.79 rain forest

Compost 11.70 6.50 59.50 28.60 11.90 43.38

DNA isolation was performed according to the instruction of suppliers for each kit. The DNA gel electrophoresis results are shown in Fig. 1 .

High molecular weight DNA was obtained by all three kits. However, as shown in Table 2 below, the DNA yield and quality were surprisingly greater with the Sox nucleic acid isolation kit, a preferred em bodiment of the present invention, for each soil sample tested.

Table 2. Extraction of soil DNA using Sox™ DNA Isolation Kit (Metagenom Bio™ Inc.)(a preferred embodiment of the present invention), PowerSoil™ DNA Isolation Kit (MO BIO™ Laboratories, Inc.) and Soil DNA Isolation Plus Kit (Norgen Biotek™ Corporation). Isolation of DNA from 0.25 gm soils was performed according to Supplier’s recommendation.

DNA yield

Soil samples DNA isolation kits A260/A280 A260/A230 (%)

Tar Sands Sox DNA Isolation Kit 1.8 1.5 100.0

PowerSoil DNA Isolation Kit 1.8 1.3 88.8

Soil DNA Isolation Plus Kit 1.9 0.7 77.1

Temperate rain forest Sox DNA Isolation Kit 1.9 1.9 100.0

PowerSoil DNA Isolation Kit 1.9 1.8 70.2

Soil DNA Isolation Plus Kit 1.9 1.5 17.3

Compost Sox DNA Isolation Kit 1.8 1.8 100.0

PowerSoil DNA Isolation Kit 1.9 1.7 84.7

Soil DNA Isolation Plus Kit 1.9 1.2 62.1

Agricultural - wheat Sox DNA Isolation Kit 1.9 1.7 100.0

PowerSoil DNA Isolation Kit 1.7 1.3 47.5

Soil DNA Isolation Plus Kit 1.9 0.6 35.4 Arctic Tundra Sox DNA Isolation Kit 1.9 1.5 100.0

PowerSoil DNA Isolation Kit 1.8 1.5 79.3

Soil DNA Isolation Plus Kit 1.8 0.7 30.7

The applicant conducted UV-visible spectra measurem ents of soil DNA preps extracted with the preferred kit of the present invention. The absorbance was m easured with a NanoDrop spectrophotom eter. As can be seen in Figure 2, the UV-visible spectra of DNA preps extracted with the preferred m ethod and kit of the present invention also indicate the high quality of isolated DNA. The strong peaks at 260 nm reflect greater DNA purity with lower contam ination with hum ic acids (peak absorbance 230 nm ) and proteins (peak absorbance 280 nm ) .

The 16S-V4 and I TS1 regions of the metagenom ic DNA isolated with : ( i) Sox™ DNA I solation Kit (MBI , Metagenom Bio™ I nc.) (a preferred embodim ent of the present invention) ; (ii) PowerSoil™ I solation Kit (MB, MO BI O™ Laboratories, I nc.) ; and (iii) Soil DNA I solation Plus Kit (N, Norgen Biotek™ Corporation) were PCR am plified, and resolved with 2% TAE agarose gel. Results are shown in Figure 3.

The amount of fungal I TS1 am plicons was greater from the DNA extracted with the preferred em bodiment of the present invention than with the other two kits. Sim ilar yields of 16S am plicons were generated with all three kits tested. As can be seen in Figure 3, the preferred method and kit of the present invention provides a highly efficient isolation and

subsequent amplification (by PCR) of target nucleic acids.

Soil com m unity profiles from DNA extracted using the preferred method and kit of the present invention are presented in Figure 4. The 16S and I TS1 profiles of 5 different soil samples indicated that various types of cells were efficiently lysed using the preferred method and kit of the present invention, resulting in inhibitor-free and contam inated-free DNA preps. The method and kit of the present invention efficiently lyses a wide variety of cell types, and produces inhibitor-free and contam inant-free DNA, suitable for downstream molecular applications, such as high- throughput sequencing.

The scope of the claims should not be lim ited by the preferred

embodim ents set forth in the exam ples, but should be given the broadest interpretation consistent with the description as a whole.

Claims

Claim s

1 . A m ethod for isolating nucleic acids from a sample comprising the steps of:

a. providing the sample;

b. lysis of biological cells in the sam ple in a reaction solution, the reaction solution com prising a detergent, a first pH buffer, a DNA desorption agent, a chelating agent, sodium chloride and silica beads; c. isolating nucleic acids and dissolved contam inants from the sam ple in a supernatant;

d. contacting the supernatant with a flocculant to precipitate and rem ove proteins;

e. contacting the supernatant with a chaotropic agent and a second pH buffer; and

f. purification of the nucleic acids by anion exchange on a spin colum n com prising a silica gel membrane.

2. The method of claim 1 , wherein the purified nucleic acids from step f are substantially free of the contam inants which inhibit am plification of the nucleic acids by polym erase chain reaction ( PCR) .

3. The method claim 2, wherein the contam inants are selected from the group consisting of polyphenols, polysaccharides, hum ic acid, enzyme inhibitors, decom posing organic matter, plant cell wall components, chitin, plant pigm ents, fulvic acid, tannin and heavy m etals.

4. The method of claim 1 , wherein the sam ple comprises one or more of soil, sediment, sand, gravel, pebbles and compost.

5. The method of claim 1 , wherein the sam ple is an environmental or process sam ple selected from the group consisting of soil, gravel, m ining tailings, sm all pebbles, ore, silt, sedim ent, glacial till, sand from slow sand filtration apparatus, com post, rhizosphere soil, used inert hydroponic m edia and coco coir.

6. The method of claim 5, wherein the used inert hydroponic m edia is selected from the group consisting of rock wool, expanded clay pebbles, perlite and verm iculite.

7. The method of claim 1 , wherein the detergent is sodium dodecyl sulfate (SDS) .

8. The method of claim 1 , wherein the first pH buffer comprises a com pound selected from the group consisting of Tris- HCI and sodium phosphate.

9. The method of claim 1 , wherein the chelating agent is

Ethylenediam inetetraacetic acid ( EDTA) .

10. The method of claim 1 , wherein the DNA desorption agent is sodium phosphate.

1 1 . The method of claim 1 , wherein the beads are glass beads having a diam eter of 0.1 m m and 0.5 m m in a ratio of about 3: 1 or a ratio within 20% variance from 3: 1 .

12. The method of claim 1 , wherein the flocculant com prises a solution of acetate and potassium in a molar ratio of about 5 Moles/ L (M) acetate: 3 Moles/ L (M) potassium or a molar ratio within 20% variance from 5 Moles/ L (M) acetate: 3 Moles/ L (M) potassium .

13. The method of claim 1 , wherein the proteins are SDS-proteins.

14. The method of claim 1 , where the chaotropic agent comprises a com pound selected from the group consisting of guanidine hydrochloride ( HCL) and guanidine (iso)thiocyanate.

15. The method of claim 1 , wherein the second pH buffer is a buffer that provides an effective pH buffering range between pH 6.0 and pH 7.0.

16. The method of claim 1 , wherein the second pH buffer is 2-(N- m orpholino) ethanesulfonic acid (MES) .

17. The method of claim 1 , wherein the nucleic acids are isolated by alcohol precipitation.

18. The method of claim 17, wherein the alcohol is ethanol.

19. The method of claim 1 , wherein the nucleic acids are purified by phenol-chloroform extraction and isolated by alcohol precipitation.

20. The method of claim 19, wherein the alcohol is ethanol.

21 . The method of any one of claim s 1 to 20, wherein the nucleic acids are DNA.

22. A nucleic acid isolation kit for conducting the method of isolating nucleic acids as defined in any one of claim s 1 to 21 , wherein the kit com prises:

(i) one or m ore bead tubes for holding silica beads;

(ii) a reaction solution comprising a detergent, a first pH buffer, a chelating agent, a desorption agent, sodium chloride and the silica beads;

(iii) a flocculant;

(iv) a chaotropic agent and a second pH buffer;

(v) one or m ore spin colum ns; and

(vi) one or m ore collection tubes.

23. A nucleic acid isolation kit comprising :

(i) one or m ore bead tubes for holding silica beads;

(ii) a reaction solution com prising a detergent, a first pH buffer, a chelating agent, a desorption agent, sodium chloride and the silica beads;

(iii) a flocculant;

(iv) a chaotropic agent and a second pH buffer;

(v) one or m ore spin colum ns; and

(vi) one or m ore collection tubes.

24. The kit of claim 23, wherein the detergent is sodium dodecyl sulfate (SDS) .

25. The kit of claim 23, wherein the first pH buffer com prises a compound selected from the group consisting of Tris- HCI and sodium phosphate.

26. The kit of claim 23, wherein the chelating agent is

Ethylenediam inetetraacetic acid ( EDTA) .

27. The kit of claim 23, wherein the DNA desorption agent is sodium phosphate.

28. The kit of claim 23, wherein the beads are glass beads having a diam eter of 0.1 m m and 0.5 m m in a ratio of about 3: 1 or a ratio within 20% variance from 3: 1 .

29. The kit of claim 23, wherein the flocculant comprises a solution of acetate and potassium in a m olar ratio of about 5 Moles/ L (M) acetate: 3 Moles/ L (M) potassium or a m olar ratio within 20% variance from 5 Moles/ L (M) acetate: 3 Moles/ L (M) potassium .

30. The kit of claim 23, where the chaotropic agent comprises a com pound selected from the group consisting of guanidine hydrochloride ( HCL) and guanidine (iso)thiocyanate.

31 . The kit of claim 23, wherein the second pH buffer is a buffer that provides an effective pH buffering range between pH 6.0 and pH 7.0.

32. The kit of claim 23, wherein the second pH buffer is 2-(N-morpholino) ethanesulfonic acid (MES) .